The long term goal of this proposal is to understand the structure, function and regulation of phospholipid biosynthesis enzymes. These studies will form a body of information which will shed light on the role of the enzymes and the phospholipids in cell function and in regulation of cell growth. The studies will be confined to Escherichia coli for which there is a large body of biochemical and genetic information already available. Regulation of phospholipid metabolism will be studied in vivo by systemically adjusting the level of phospholipid biosynthetic enzymes using extrachromosomal copies of their structural genes under exogenous regulation. The rates of synthesis at several points in the biosynthetic pathway will be determined using substrate analogues which lead to the synthesis of phospholipids which are not further metabolized. In this way the pattern of coordination between synthesis and turnover at several points in the pathway will be established. The importance of phospholipid composition to cell physiology will be assessed by varying composition through altering the level of biosynthetic enzymes. The role of specific phospholipids in cell function will be studied by complete inactivation of genes which code for the synthesis of central enzymes in phospholipid metabolism. Study of cell phenotype and supplementation by exogenously added phospholipid will be used to assess the importance of the phospholipid and its role as a precursor to other cellular components. The genetic regulation of the synthesis of phospholipid biosynthetic enzymes will be studied by characterizing the pssR gene product which acts in a trans manner in regulating the synthesis of the phosphatidylserine synthase. Finally, studies on the synthesis and assembly of phospholipid biosynthetic enzymes will focus on the mechanism of formation of the pyruvate prosthetic group of the phosphatidylserine decarboxylase and the role of the membrane in the posttranslational processing of this protein.